We reported recently that human and mouse diabetic retina have increased iron accumulation, and inducing diabetes in a genetic mouse model of systemic iron overload resulted in accelerated progression of diabetic retinopathy (DR). Iron, although an essential nutrient, when accumulated excessively leads to tissue damage. However, systemic administration of iron chelators is not a feasible therapeutic option to reduce tissue iron levels due to potential side effects of lowering serum iron levels. New mechanistic insights into the role of iron in ketone body synthesis and utilization is a critical gap in knowledge addressed in this application facilitating new therapeutic targets for DR. During conditions of prolonged fasting and diabetes, body utilizes fatty acids to make ketone bodies as an alternate metabolic fuel. ?- hydroxybutyrate (?-OHB), acetoacetate and acetone, the three ketones produced in the body, are metabolically important because their accumulation in blood can cause ketoacidosis and secondly, depending on the physiological state, ketones supply energy for cell survival. The central hypothesis of this proposal is that retinal iron accumulation during DR inhibits endogenous ketone ?- OHB production, activating NLRP3 inflammasome signaling and thereby accelerating cell death. We will test our hypothesis using three distinct conceptual, computational and experimental strategies. In aim 1, we will explore how cellular iron accumulation modulates endogenous ketone body synthesis. Liver, colon and retina synthesize most of the endogenous ketones. Here, we propose to determine the effect of iron on the function of the mitochondrial enzyme Bdh1, which catalyzes the final reaction for producing ?-OHB, the principal ketone in circulation using 2 novel mouse lines, constitutive and RPE-specific Bdh1 knockout mice. In aim 2, we will examine the mechanisms by which iron-associated decrease in ketone body ?-OHB impacts inflammation during DR. Our lab and others have reported that retinal iron increases NLRP3 inflammasome. A report in Nature Medicine showed that exogenous ?-OHB, but not the ketone acetoacetate, reduces NLRP3 inflammasome. Here we propose to investigate if iron associated reduction in endogenous ?-OHB synthesis augments inflammation through histone deacetylase-Foxo3a signaling. In aim 3, we will explore the effect of iron on the uptake of hepatic/RPE produced ?-OHB by the neuronal cells, which are primarily dependent on ketone bodies during conditions of low glucose availability. We previously reported that monocarboxylate transporter SMCT1, the only known ?-OHB transporter in ganglion cells, is downregulated during iron overload in retina. We will analyze the epigenetic mechanisms by which iron alters cellular uptake of ?-OHB in neuronal cells causing cell death. The three proposed aims are supported by the past 10 years of training in iron homeostasis, a vibrant research environment at SLU, and continuing professional guidance and collaboration with senior faculty. Successful completion of this project will advance our understanding of how iron alters ketone metabolism and drive the field into new and underexplored areas, like metabolic reprogramming for efficient ketone body utilization.